Reovirus Neuropathogenesis

呼肠孤病毒神经发病机制

• 批准号: 10607594
• 负责人: TERENCE S. DERMODY
• 金额: $ 56.55万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-22 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607594
• 关键词: 3-Dimensional; Adult; Afferent Neurons; Atomic Force Microscopy; Axon; Axonal Transport; Binding; Biophysics; Brain; Brain imaging; CRISPR-mediated transcriptional activation; Capsid; Capsid Proteins; Cells; Cerebellum; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Cryoelectron Microscopy; Cultured Cells; Development; Disease; Double Stranded RNA Virus; Dynein ATPase; Electron Microscopy; Encephalitis; Engineering; Fiber; Genome; Hippocampus (Brain); Human; Immunocompetent; Immunoglobulins; In Vitro; Infection; Integration Host Factors; Integrins; Interneurons; Intervention; Intramuscular; Knockout Mice; Knowledge; Malignant Neoplasms; Mammals; Mediating; Morbidity - disease rate; Motor; Mus; Mutagenesis; Nervous system structure; Neuraxis; Neurons; Neuropathogenesis; Neurotropism; Newborn Infant; Oncolytic; Pattern; Peripheral; Pharmacology; Population; Process; Proteins; Reovirus; Reovirus Infections; Research; Resolution; Role; Route; Sensory Receptors; Series; Serotyping; Structure; Synapses; System; Testing; Thalamic structure; Tissues; Tropism; Viral; Viral Encephalitis; Viral Pathogenesis; Virulence; Virus; Work; blind; cell type; clinical development; experimental study; fast axonal transport; in vivo; mortality; mutant; nervous system disorder; neural circuit; neuronal cell body; neurotropic; neurotropic virus; neurovirulence; new therapeutic target; oncolytic vector; particle; receptor; receptor function; reconstruction; recruit; relating to nervous system; reverse genetics; screening; targeted treatment; transmission process; uptake; virus host interaction

Viral encephalitis is an important cause of morbidity and mortality in children and adults. Key knowledge gaps about the pathogenesis of viral encephalitis include how neurotropic viruses target the central nervous system (CNS), internalize into neurons, and disseminate in the brain. The proposed research uses reovirus, a genetically tractable double-stranded RNA virus that causes encephalitis and shows promise as an oncolytic agent, to dissect mechanisms of viral tropism, cell entry, and spread in the CNS. Reovirus strains display serotype-specific patterns of systemic dissemination and neurologic disease. Serotype 3 (T3) reoviruses are exquisitely neurotropic, internalize into neurons using macropinocytosis, and spread intracellularly by fast axonal transport. Reovirus uses β1 integrin to internalize into some types of cells, but its function in neural cell entry is unknown. Other previously identified reovirus receptors are dispensable for reovirus infection in the murine CNS. We recently identified a new reovirus receptor, paired immunoglobulin-like receptor B (PirB), that allows serotype-independent reovirus infection of cultured cells but is required for efficient T3 infection of neurons and full neurovirulence. Three integrated specific aims are proposed to enhance knowledge of reovirus neuropathogenesis that may have broader applications to other neurotropic viruses. In Specific Aim 1, functions of host receptors in reovirus neuropathogenesis will be determined. Biophysical interactions between PirB and reovirus will be assessed using cryo-electron microscopy, atomic-force microscopy, and structure- guided mutagenesis. The function of PirB in reovirus disease will be elucidated by comparing infection and virulence in wild-type (WT) and PirB-null mice. T3 reovirus-specific receptors that couple with PirB for neurotropism will be identified using CRISPR activation screening. In Specific Aim 2, mechanisms of reovirus entry into CNS neurons will be defined. The functions of PirB and β1 integrin in reovirus neural entry will be determined using mutant receptors and receptor-blind viruses. Receptor-dependent control of macropinocytosis will be elucidated using super-resolution, live-cell, and electron microscopy. Mechanisms of dynein motor recruitment for reovirus transport within axons will be dissected by investigating interactions between WT and mutant reovirus receptors and dynein subunits. In Specific Aim 3, the basis of reovirus dissemination in the CNS will be elucidated. The role of host receptors in reovirus neural transit will be defined using whole-brain imaging and three-dimensional reconstruction to trace reovirus infection and neural spread in WT and receptor- null mice. Mechanisms of reovirus transsynaptic transmission will be elucidated using in vitro and in vivo neural circuits. The effect of altering synaptic activity on reovirus neural transit will be tested using pharmacologic intervention and chemogenetically altered mice. Taken together, these studies will define mechanisms used by neurotropic reovirus to infect and disseminate in the brain and may promote further development of reovirus oncolytics that selectively target the nervous system.

病毒性脑炎是儿童和成人发病和死亡的重要原因。关键知识差距 关于病毒性脑炎的发病机制，包括嗜神经病毒如何靶向中枢神经系统 （中枢神经系统），内化到神经元中，并在大脑中传播。拟议的研究使用呼肠孤病毒，一种 遗传上易于控制的双链 RNA 病毒，可引起脑炎并显示出作为溶瘤药物的前景 剂，剖析病毒向性、细胞进入和中枢神经系统传播的机制。呼肠孤病毒株展示 全身传播和神经系统疾病的血清型特异性模式。血清型 3 (T3) 呼肠孤病毒是 精致的神经营养性，通过巨胞饮作用内化到神经元中，并快速在细胞内传播 轴突运输。呼肠孤病毒使用β1整合素内化到某些类型的细胞中，但其在神经细胞中的功能 条目未知。其他先前发现的呼肠孤病毒受体对于呼肠孤病毒感染来说是可有可无的。 小鼠中枢神经系统。我们最近发现了一种新的呼肠孤病毒受体，配对免疫球蛋白样受体 B (PirB)， 允许对培养细胞进行血清型独立的呼肠孤病毒感染，但对于有效的 T3 感染是必需的 神经元和完整的神经毒力。提出了三个综合具体目标以增强呼肠孤病毒的知识 神经发病机制可能对其他嗜神经病毒有更广泛的应用。在具体目标 1 中， 将确定宿主受体在呼肠孤病毒神经发病机制中的功能。之间的生物物理相互作用 PirB 和呼肠孤病毒将使用冷冻电子显微镜、原子力显微镜和结构显微镜进行评估 引导诱变。通过比较感染和呼肠孤病毒疾病，将阐明 PirB 在呼肠孤病毒疾病中的功能。 野生型 (WT) 和 PirB 缺失小鼠的毒力。 T3 呼肠孤病毒特异性受体与 PirB 偶联 将使用 CRISPR 激活筛选来鉴定向神经性。在具体目标 2 中，呼肠孤病毒的机制 进入中枢神经系统神经元的入口将被定义。 PirB 和 β1 整合素在呼肠孤病毒神经进入中的功能将是 使用突变受体和受体盲病毒确定。巨胞饮作用的受体依赖性控制 将使用超分辨率、活细胞和电子显微镜来阐明。动力蛋白马达的机制 将通过调查 WT 和 WT 之间的相互作用来剖析呼肠孤病毒在轴突内运输的招募 突变呼肠孤病毒受体和动力蛋白亚基。在具体目标 3 中，呼肠孤病毒在 CNS将被阐明。宿主受体在呼肠孤病毒神经传递中的作用将使用全脑来定义 成像和三维重建以追踪 WT 和受体中呼肠孤病毒感染和神经传播 无效小鼠。将利用体外和体内神经网络阐明呼肠孤病毒跨突触传播的机制 电路。将使用药理学方法测试改变突触活性对呼肠孤病毒神经传递的影响 干预和化学遗传学改变的小鼠。总的来说，这些研究将定义所使用的机制 嗜神经性呼肠孤病毒在大脑中感染和传播，并可能促进呼肠孤病毒的进一步发展 选择性靶向神经系统的溶瘤药物。